Functional inactivation (by mutation) of the cystic fibrosis transmembrane conductance regulator (CFTR), a small conductance, cAMP activated chloride ion channel, leads to developmental defects that cause male infertility in humans. The mechanism by which inactivation of the CFTR gene causes infertility is poorly understood but is believed to be due in part to malformations or pathological changes in the male genital duct system. The goals of the proposed research are to decipher the transcriptional networks that regulate luminal environment in the epididymis, by building on our current understanding of the mechanisms regulating expression of the CFTR gene. Mutations in CFTR cause the devastating inherited disorder cystic fibrosis (CF). The CFTR channel makes an essential contribution to ion transport and the luminal environment in the epididymis and its loss causes obstruction and/or loss of the male genital ducts and subsequent infertility. The epididymis forms the proximal part of the excretory duct system of the male reproductive tract and is a complex organ that secretes and absorbs ions, water and inorganic solutes. Critically, it provides the correct luminal environment for normal sperm maturation. In normal epididymis the luminal environment is established and maintained through the functional cooperation of multiple ion channels, ion exchangers, solute carriers and transporters, though the mechanisms that coordinate the expression of these genes has not been well studied. In the first specific aim we will determine how modifications in the chromatin structure of the CFTR locus control its expression in the epididymis. We will evaluate the cis-acting enhancers and insulator elements that likely establish the looped conformation of the active locus and determine how trans-acting factors such as CTCF and cohesin influence this spatial organization. Moreover, we will investigate mechanisms and DNA- binding proteins that may suppress gene expression in cells where CFTR is less active or inactive. Experiments in the second specific aim will determine the transcriptional pathways that coordinate expression of ion channels, ion exchangers, transporters and solute carriers that interact with CFTR in the epididymis epithelium to establish and maintain the luminal environment. We will use a genome-wide approach (DNase- seq) to identify the cis-acting regulatory elements for genes encoding the proteins that contribute to ion and solute transport across the epididymal epithelium. Following mapping of these elements we will use combined bioinformatic and experimental approaches to identify the trans-acting factors that mediate the coordinated regulation of gene expression. Successful conduct of these studies will provide unique translational opportunities to modify male fertility. In disease states we may be able to reinstate the normal epididymal luminal environment and thereby restore fertility. Through modulation of this environment we may also be able to inhibit normal sperm maturation and thereby develop novel methods of birth control.